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选择含有反向筛选基因sacB标记的pRE112质粒为自杀载体,利用等位基因交换的方法成功敲除了布鲁氏菌弱毒疫苗S19株、S2株、M5株的bp26基因,构建了具有非抗性基因标记的缺失株S19-Δbp26、S2-Δbp26、M5-Δbp26。将构建的重组自杀质粒pRE-Δbp26(缺失了bp26基因的681个碱基)电转化入布鲁氏菌后,经过5μg/mL氯霉素筛选单交换子和70g/L蔗糖筛选同源重组双交换子,用菌落PCR及DNA测序的方法进行验证。结果表明,bp26基因的缺失改造成功,连续传20代后菌落PCR及DNA测序的结果显示突变株具有遗传稳定性。以pRE112自杀质粒为基础构建无抗性基因标记的布鲁氏菌缺失株为布鲁氏菌的基因功能研究奠定了基础,同时Δbp26缺失株的构建可为新型布鲁氏菌疫苗的研制奠定基础。
The plasmid pRE112 containing the reverse selection gene sacB was selected as the suicide vector. The allelic gene swap method was used to successfully knock out the bp26 gene of Brucella attenuated vaccine S19, S2 and M5 and construct the non-resistance gene Marked deletion strains S19-Δbp26, S2-Δbp26, M5-Δbp26. The constructed recombinant suicide plasmid pRE-Δbp26 (bp1 68 bp) was transformed into Brucella and screened by homologous recombination double crossover with 5 μg / mL chloramphenicol single crossover and 70 g / Exchanges were verified by colony PCR and DNA sequencing. The results showed that the deletion of bp26 gene was successfully transformed, and the results of colony PCR and DNA sequencing showed that the mutant strains had genetic stability. The construction of the gene-resistant Brucella null strain based on the suicide plasmid pRE112 lays the foundation for the gene function research of Brucella and the construction of the Δbp26 deletion lays the foundation for the development of a new Brucella vaccine .